The development of a flow cytometric intracellular assay for the presence of the proteins gp91phox and p47phox has been accomplished using specific cell fixation and permeabilization procedures. This method has been applied to evaluate granulocytes from chronic granulomatous disease (CGD) patients and a pattern has been observed that distinguishes the X-linked form of CGD (gp91 phox deficient) from the most common autosomal recessive form of CGD (p47phox deficient). In addition, this method confirms that the mater-nal carriers of the X- linked form have two populations of circulating granulocytes, one normal (normal gp91phox expression) and one abnormal (absent gp91phox expression). The combina-tion of this method with the DHR assay allows for a rapid and accurate genotype assignment in the majority of CGD patients. Additional monoclonal antibodies are under development directed at native p47 phox, p22 phox, and p67 phox. These reagents should provide for the accurate genotyping of CGD by flow cytometry.